Increased radiosensitivity and radiothermosensitivity of human pancreatic MIAPaCa-2 and U251 glioblastoma cell lines treated with the novel Hsp90 inhibitorNVP-HSP990
© Milanović et al.; licensee BioMed Central Ltd. 2013
Received: 14 September 2012
Accepted: 26 February 2013
Published: 28 February 2013
Background and purpose
Heat shock Protein 90 (Hsp90) is a molecular chaperone that folds,stabilizes, and functionally regulates many cellular proteins involved inoncogenic signaling and in the regulation of radiosensitivity. It isupregulated in response to stress such a heat. Hyperthermia is a potentradiosensitizer, but induction of Hsp90 may potentially limit its efficacy.Our aim was to investigate whether the new Hsp90 inhibitor NVP-HSP990increases radiosensitivity, thermosensitivity and radiothermosensitivity ofhuman tumor cell lines.
Material and methods
U251 glioblastoma and MIA PaCa-2 pancreatic carcinoma cells were used. Todetermine clonogenic survival, colony forming assays were performed. Cellviability and proliferation were assesed by Trypan blue staining. Cell cycleand apoptosis analyses were performed by flow cytometry. DAPI staining wasused to detect mitotic catastrophe.
NVP- HSP990 increased the thermosensitivity, radiosensitivity andradio-thermosensitivity of both cell lines in clonogenic assays.72 hours after irradiation with 4 Gy, a significant reduction incell number associated with considerable G2/M acumulation and mitoticcatastrophe as well as cell death by apoptosis/necrosis was observed.
Treatment with NVP- HSP990 strongly sensitized U251 and MIA PaCa-2cells to hyperthermia and ionizing radiation or combination thereof throughaugmentation of G2/M arrest, mitotic catastrophe and associatedapoptosis.
KeywordsRadiosensitivity Radiothermosensitivity NVP-HSP990
Heat shock protein 90 (Hsp90) is an evolutionary conserved molecular chaperone whichunder physiological conditions participates in protein folding, intracellulartransport, maintenance and degradation of proteins. Proteins, which are activatedand stabilized by Hsp90, are referred to as “clients”. A lot of them arecrucial for constitutive cell signaling and adaptive responses to stress . Hsp90 is expressed at 2–10-fold higher levels in tumor tissue thanin normal tissue . Its most important function is to protect mutated and overexpressedoncoproteins from misfolding and degradation. It has been recognized to be essentialfor the stability and function of a wide variety of kinases such as EGFR, Erb-B2,Akt, BCR-ABL, VEGFR2 involved in cell cycle regulation, survival and oncogenicsignaling . These proteins play also critical roles in the regulation ofradiosensitivity [4–6]. Thus, the inhibition of Hsp90 may represent an attractive therapeuticstrategy not only reducing basal survival of tumor cells but also increasing theirradiosensitivity.
Heat is a very potent radiosensitizer in vitro and in vivo[7, 8]. Clinical studies have shown that the combination of conventionalradiation therapy with hyperthermia leads to significantly better tumor control . Hsp90 is heat-inducible in normal and in tumor cells . This heat induction of Hsp90 could limit desirable effects of ionizingradiation (IR) via activation of “client proteins” which may contributeto radioresistance as mentioned above. Theoretically, small molecules designed toinhibit Hsp90 might increase the effect of hyperthermia followed by enhancedradiosensitivity.
NVP-HSP990 is a novel, highly potent orally available 2-aminothienopyrimidine class,non-geldanamycin based Hsp90 inhibitor . It has been shown that treatment of established cell lines fromdifferent tumor entities with the non- geldanamycin based Hsp90 inhibitorsNVP-BEP800 and NVP-AUY922 increase their sensitivity towards IR .
Glioblastoma multiforme and pancreatic carcinoma represent tumors which are resistantto conventional radiochemotherapeutical regimes and despite intensive research anddevelopment of new targeted therapies prognosis of patients with these tumorsremains poor [13, 14], indicating the need for new therapeutic approaches.
Here we investigated whether treatment with the Hsp90 inhibitor NVP-HSP990 andhyperthermia enhance thermal sensitivity and consequently radiosensitivity of U251glioma and MIA PaCa-2 pancreatic carcinoma cells.
Materials and methods
Cell culture and reagents
The U251 human glioblastoma and pancreatic carcinoma MIA PaCa-2 cell line wereobtained from the tumor bank of the National Cancer Institute (NCI), Frederick,Maryland. Cells were grown as a monolayer in RPMI-1640 culture mediumsupplemented with 10% foetal bovine serum (FBS; Biochrom, Berlin, Germany) at37°C under 8.5% CO2. NVP-HSP990 was kindly provided by NovartisInstitutes for Biomedical Research (Basel, Switzerland). The drug was dissolvedin DMSO and stock solutions were stored at −20°C.
Hyperthermia was provided by a cell incubator (Heraeus, type Heracell), reachingtemperatures of up to ~55°C with a precision of 0.1°C. The temperatureof 37°C was chosen as control. Before starting a hyperthermia, thetemperature was controlled with 3 thermocouples from the interstitialhyperthermia device (Academic Ziekenhuis Utrecht, The Netherlands), which wereinserted in needles and placed in the cell culture flasks filled with 5 mlculture media. Each thermocouple has 7 different points from which thetemperature was measured. When the average temperature value in the incubatorreached 42°C, the cells were incubated for 1 hour and afterwardsreplaced to 37°C.
Hyperthermia and drug treatment
To investigate the effect of NVP-HSP990 on colony formation as a sole compound,cells were treated with increasing drug concentrations (0.01, 0.02, 0.05 and0.1 μM). In combination treatment with hyperthermia, the cells weretreated with the same drug concentrations. Immediately after adding the drug,the cells were incubated for 1 hour at 42°C and afterwards replacedto 37°C. 24 hours later, the cells were trypsinised and 100 cellswere plated for colony forming assay (CFA) without drug using25 cm2 tissue flasks (Falcon Becton-Dickinson,Germany).
Irradiation was performed at room temperature using a Gammacell 40137Cs laboratory irradiator. After irradiation, the cells wererecovered in growth medium for 24 hours until harvest.
Combined treatment with NVP-HSP990, hyperthemia and IR
Colony forming assay (CFA)
After allowing the cells to attach to the petri dish, the cells were irradiatedwith 0, 2, 4, 6 or 8 Gy without NVP-HSP990. 12 days after seeding,the colonies were fixed with methanol/acetic acid (3:1) and stained with crystalviolet dye (1%). The number of colonies containing at least 50 cells wasdetermined, and the surviving fractions were calculated. The curves werenormalised to SF1 (100% cell survival). The surviving fractions were calculatedusing the plating efficiency for each treatment group (42°C, NVP-HSP990 or42°C + NVP-HSP990 combination). Plating efficiency andsurviving fractions were determined for each cell line and treatment. Cellsurvival curves were fitted by the linear-quadratic modelSF = exp[−(αD + βD2)].
Assessment of cell proliferation and viability by Trypan blue exclusion andFACS analysis
To determine the number of viable cells, trypan blue exclusion tests wereconducted. To assess induction of apoptosis and global cell death, annexin-V andpropidium-iodide (PI) double staining was performed using the Annexin-VApoptosis Detection Kit (Miltenyi Biotec). Briefly, U251 and MIA PaCa-2 cellswere treated with 0.05 μM NVP-HSP990. Immediately after adding thedrug, the cells were incubated for 1 hour at 42°C and then replacedto 37°C. 24 hours later, the growth medium was replaced and the cellswere irradiated with a single dose of 4 Gy. 24 and 72 hours later,the cells were stained with annexin V-FITC and PI and analyzed by FACS (FMT 500)from Beckman Coulter.
Cell cycle analyses
Exponentially growing U251 and MIA PaCa-2 cells were treated and fixed 8, 24 and48 hours later with 70% ethanol. After storage at −20°Covernight, the cells were washed and incubated with PI (50 μg/mL) andRNase (100 μg/mL) for 2 h at 4°C. After washing, the cellswere analyzed for DNA content by flow cytometry.
Assessment of mitotic catastrophe
U251 cells were treated as described. 3 or 5 days later, the cells werefixed, stained with 4'-6-diamidino-2-phenylindole (DAPI) and analysed under anOlympus BX41 fluorescence microscope equipped with a digital camera CC-12 softimaging system (U-CMAD3, Olympus). For each assessment of the extent of mitoticcatastrophe, 200 nuclei were examined.
The Mann–Whitney U Test and Kruskal-Wallis analysis of variancewere used to compare quantification data. Statistical analysis was conductedwith Statistical Package for Social Sciences software (SPSS Inc.). We used a2-sided test with significance level of 0.05 for all statistical analyses.Synergy was calculated by the fractional product method that allows anevaluation of synergy at a defined level of effect .
The effect of hyperthermia and NVP-HSP990 on clonogenic survival of U251 andMIA PaCa-2 cells
The effect of NVP-HSP990 on cellular radiosensitivity assesed by CFA
Combined treatment with NVP-HSP990 and hyperthermia strongly increases theradiosensitivity of U251 and MIA PaCa-2 cells
The influence of the combined treatment with NVP-HSP990 and hyperthermia on theradiosensitivity of both cell lines was also analysed by CFA. While treatmentwith 0.05 μM NVP-HSP990 or heating with 42°C for 1 hourhad only a modest effect on radiosensitivity of U251 cells (Figure 3C), the combination treatment caused a potentradiosensitization. In case of irradiation with 6 or 8 Gy, no colonyformation was detected anymore. Treatment of the MIA PaCa-2 cells (Figure 3D) with 0.05 μM NVP-HSP990 had a more pronouncedradiosensitizing effect in comparison to U251 cells. Heating of these cellscaused further radiosensitization at any irradiation dose. At a dose of8 Gy, no colony formation was observed anymore.
Effect of treatments on proliferation and apoptosis in U251 and MIA PaCa-2cells
Cell cycle alterations in U251 and MIA PaCa-2 cells
Effect on mitotic catastrophe in U251 cells
The treatmant of patients with glioblastoma and pancreatic carcinoma remains achallenge. In the present study, we found that pretreatment with the novel Hsp90inhibitor NVP-HSP990 strongly sensitizes U251 glioma and MIA PaCa-2 pancreaticcarcinoma cells to hyperthermia and IR and particulary to the combination thereof.The triple combination caused a significant reduction in cell number which wasassociated with a morphological alterations typical of mitotic catastrophe andapoptosis.
There is a lot of experimental [16–18] and clinical evidence that hyperthermia can increase the effectiveness ofother conventional treatments such chemotherapy  or especially radiotherapy [20, 21]. The exact mechanism how hyperthermia increases radiosensitivity is stillnot completely elucidated but it has been proposed that hyperthermia may interferewith radiation-induced DNA damage . On the other hand, heat shock proteins mediate resistance tohyperthermia . It has been shown that inhibition of Hsp90 with geldanamycin causesdelayed and impaired recovery from heat shock . In HEK293 cells, specific inhibition of Hsp90 together with short termexposure (20–60 min) to 42°C was highly cytotoxic, throughaccelerated degradation of Cdc25A , a member of the CDC25 family of phosphatases which is specificallydegraded in response to DNA damage . These findings support the hypothesis that the antineoplastic effect ofhyperthermia could be potentiated by concurrent inhibition of Hsp90. In ourexperimental conditions, we observed that U251 and MIA PaCa-2 cells which wereincubated for 1 hour at 42°C and concurrently treated with NVP-HSP990showed a significantly lower capability to form colonies in comparison to cellswhich were treated with only one of the two modalities.
The effects of combined hyperthermia and irradiation treatment depend on manydifferent factors such a heating temperature, heating time, sequence and timeinterval between the two modalities . Despite intensive research, it is still not clear wheather hyperthermiabefore or after irradiation causes a more pronounced enhancement of radiationdamage. Probably, this effect is cell-type specific. In the case of concurrentirradiation and hyperthermia, maximal additive/synergistic effects can be expectedwhile increasing the intervals of time between hyperthermia and IR, regardless ofsequence, will abrogate the radiosensitisation induced by hyperthermia . As expected, because of the time interval of 23 hours betweenhyperthermia and IR, we observed only a weak influence of hyperthermia onradiosensitivity, cell cycle distribution, induction of mitotic catastrophe andapoptosis.
It has been proposed that the radiosensitising effect of Hsp90 inhibitors is causedby degradation of several proteins such a ErbB2, EGFR, Raf-1 and Akt [29, 30] which reportedly can influence radioresistance. DNA repair and cell cyclecheckpoint activation are other proposed mechanisms by which Hsp90 can influence theDNA damage response to IR . A previous study reported that a 24 h-pretreatment with an Hsp 90inhibitor similar to NVP-HSP990, NVP-BEP800, caused an increase in radiosensitivityin two glioblastoma, one lung carcinoma and one fibrosarcoma cell line throughcell-cycle impairment, increased DNA damage and repair protraction . The authors found that changes in the expression of survival markers(Hsp90, Hsp70, Akt, phospho-Akt, Raf-1 and survivin), an apoptosis-associatedprotein (cleaved caspase 3) or of the functional p53 status did not significantlycontribute to the sensitivity of two out of four tested cell lines to NVP-BEP800alone or in combination with IR. Another group found that the geldanamycin-basedHsp90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG)enhances radiosensitization of human U251 and MIA PaCa-2 cells . The treatment of the cells with this compound caused a reduction of theexpression of Akt, Raf-1 and especially ErbB2. The authors further reported thattreatment of DU145 prostate carcinoma cells with 17-DMAG abrogated the G2- andS-phase cell cycle checkpoints and enhanced the radiosensitivity of the cells.
It has been reported that treatment of the human lung adenocarcinoma cell line A549with KNK437, an benzylidene lactam compound which acts as a heat shock proteininhibitor, causes the enhancement of thermal radiosensitization in mild hyperthermiacombined with low dose IR . In the same study, it has been demonstrated that KNK437 causedinhibition of Hsp72 and Hsp27 expression. NVP-HSP990 shows a different mechanism ofaction; it binds to the NH2-terminal ATP-binding pocket of Hsp90 while KNK437inhibits synthesis of various heat shock proteins at the mRNA level. KNK437 has alsobeen proposed to induce radioresistance of A-172 human glioblastoma and humansquamous cell carcinoma cells .
In our experiments, we observed that pretreatment of U251 cells with NVP-HSP990 andhyperthermia before irradiation with 4 Gy caused a delayed acumulation ofcells in the G2/M phase. The strongest effect was detected 24 hours afterirradiation. Three to five days after the irradiation teatment, we observed a strongincrease of the number of cells with morphological signs of mitotic catastrophe(micro- and multinucleated cells) [34, 35]. The number of apoptotic cells also increased. As large numbers ofnecrotic cells (taking up PI) were not found, this suggests that mitotic catastropheconstitutes a prelude to apoptotic cell death. Similar findings were reported for anovel small molecule inhibitor that lowers the threshold for Hsf1 (Heat shock factorprotein 1) activation . The inhibition enhanced thermal sensitivity and significant thermalradiosensitization followed by loss of mitochondrial potential and mitoticcatastrophe in HT29 colon carcinoma cells.
Taken together, our study shows that NVP-HSP990, a fully synthetic, orally availableHsp90 inhibitor exibits strong anti-tumor effects on U251 human glioblastoma and MIAPaCa-2 pancreatic carcinoma cells through an increase of sensitivity towards heatand ionising irradiation. Further preclinical studies are warranted to clarify thecomplex mechanisms of its action and to explore the therapeutic potential of thisapproach in vivo.
- Wandinger SK, Richter K, Buchner J: The Hsp90 chaperone machinery. J Biol Chem 2008, 283: 18473-18477. 10.1074/jbc.R800007200View ArticlePubMed
- Gooljarsingh LT, Fernandes C, Yan K: A biochemical rationale for the anticancer effects of Hsp90 inhibitors: slow,tight binding inhibition by geldanamycin and its analogues. Proc Natl Acad Sci U S A 2006, 103: 7625-7630. 10.1073/pnas.0602650103View ArticlePubMedPubMed Central
- Trepel J, Mollapour M, Giaccone G, Neckers L: Targeting the dynamic HSP90 complex in cancer. Nat Rev Cancer 2010, 10: 537-549.View ArticlePubMed
- Chautard E, Loubeau G, Tchirkov A, Chassagne J, Vermot-Desroches C, Morel L, Verrelle P: Akt signaling pathway: a target for radiosensitizing human malignantglioma. Neuro Oncol 2010, 12: 434-443.PubMedPubMed Central
- Pietras RJ, Poen JC, Gallardo D, Wongvipat PN, Lee HJ, Slamon DJ: Monoclonal antibody to HER-2/neureceptor modulates repair ofradiation-induced DNA damage and enhances radiosensitivity of human breastcancer cells overexpressing this oncogene. Cancer Res 1999, 59: 1347-1355.PubMed
- Pirollo KF, Hao Z, Rait A, Ho CW, Chang EH: Evidence supporting a signal transduction pathway leading to theradiation-resistant phenotype in human tumor cells. Biochem Biophys Res Commun 1997, 230: 196-201. 10.1006/bbrc.1996.5922View ArticlePubMed
- Kampinga HH, Dikomey E: Hyperthermic radiosensitization: mode of action and clinical relevance. Int J Radiat Biol 2001, 77: 399-408. 10.1080/09553000010024687View ArticlePubMed
- Frey B, Weiss EM, Rubner Y: Old and new facts about hyperthermia-induced modulations of the immunesystem. Int J Hyperthermia 2012, 28: 528-542. 10.3109/02656736.2012.677933View ArticlePubMed
- Horsman MR, Overgaard J: Hyperthermia: a potent enhancer of radiotherapy. Clin Oncol 2007, 19: 418-426. 10.1016/j.clon.2007.03.015View Article
- Ciocca DR, Fuqua SA, Lock-Lim S, Toft DO, Welch WJ, McGuire WL: Response of human breast cancer cells to heat shock and chemotherapeuticdrugs. Cancer Res 1992, 52: 3648-3654.PubMed
- Massey AJ, Schoepfer J, Brough PA, Brueggen J: Preclinical antitumor activity of the orally available heat shock protein 90inhibitor NVP-BEP800. Mol Cancer Ther 2010, 9: 906-919. 10.1158/1535-7163.MCT-10-0055View ArticlePubMed
- Stingl L, Stühmer T, Chatterjee M, Jensen MR, Flentje M, Djuzenova CS: Novel HSP90 inhibitors, NVP-AUY922 and NVP-BEP800, radiosensitise tumourcells through cell-cycle impairment, increased DNA damage and repairprotraction. Br J Cancer 2010, 25: 1578-1591.View Article
- Stupp R, Mason WP, van den Bent MJ: Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005, 352: 987-996. 10.1056/NEJMoa043330View ArticlePubMed
- Conroy T, Desseigne F, Ychou M: FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer. N Engl J Med 2011, 364: 1817-1825. 10.1056/NEJMoa1011923View ArticlePubMed
- Webb JL (Ed): Effect of more than one inhibitor; enzyme and metabolic inhibitors. Vol.1. Academic Press: New York; 1963:466-487.
- Li GC, Kal HB: Effect of hyperthermia on the radiation response of two mammalian celllines. Eur J Cancer 1977, 13: 65-69.View ArticlePubMed
- Krawczyk PM, Eppink B, Essers J: Mild hyperthermia inhibits homologous recombination, induces BRCA2degradation, and sensitizes cancer cells to poly (ADP-ribose) polymerase-1inhibition. Proc Natl Acad Sci U S A 2011, 108: 9851-9856. 10.1073/pnas.1101053108View ArticlePubMedPubMed Central
- Bergs JW, Franken NA, Haveman J: Hyperthermia, cisplatin and radiation trimodality treatment: a promisingcancer treatment? A review from preclinical studies to clinicalapplication. Int J Hyperthermia 2007, 23: 329-341. 10.1080/02656730701378684View ArticlePubMed
- Wendtner CM, Abdel-Rahman S, Krych M: Response to neoadjuvant chemotherapy combined with regional hyperthermiapredicts long-term survival for adult patients with retroperitoneal andvisceral high-risk soft tissue sarcomas. J Clin Oncol 2002,20(14):3156-3164. 10.1200/JCO.2002.07.146View ArticlePubMed
- Lutgens L, van der Zee J, Pijls-Johannesma M: Combined use of hyperthermia and radiation therapy for treating locallyadvanced cervical carcinoma. Cochrane Database Syst Rev 2010, 1: CD006377.PubMed
- Overgaard J, Gonzalez Gonzalez D, Hulshof MC: Randomised trial of hyperthermia as adjuvant to radiotherapy for recurrent ormetastatic malignant melanoma. European Society for HyperthermicOncology. Lancet 1995, 345: 540-543. 10.1016/S0140-6736(95)90463-8View ArticlePubMed
- Kampinga HH: Cell biological effects of hyperthermia alone or combined with radiation ordrugs: a short introduction to newcomers in the field. Int J Hyperthermia 2006, 22: 191-196. 10.1080/02656730500532028View ArticlePubMed
- Calderwood SK, Ciocca DR: Heat shock proteins: stress proteins with Janus-like properties in cancer. Int J Hyperthermia 2008, 24: 31-39. 10.1080/02656730701858305View ArticlePubMed
- Madlener S, Rosner M, Krieger S: Short 42 degrees C heat shock induces phosphorylation and degradation ofCdc25A which depends on p38MAPK, Chk2 and 14.3.3. Hum Mol Genet 2009, 18: 1990-2000. 10.1093/hmg/ddp123View ArticlePubMed
- Xiao Z, Chen Z, Gunasekera AH: Chk1 mediates S and G2 arrests through Cdc25A degradation in response toDNA-damaging agents. J Biol Chem 2003, 278: 21767-21773. 10.1074/jbc.M300229200View ArticlePubMed
- Hahn GM: Hyperthermia and cancer. New York: Plenum Press; 1982.View Article
- Sapareto SA, Raaphorst GP, Dewey WC: Cell killing and the sequencing of hyperthermia and radiation. Int J Radiat Oncol Biol Phys 1979, 5: 343-347. 10.1016/0360-3016(79)91214-8View ArticlePubMed
- Bisht KS, Bradbury CM, Mattson D: Geldanamycin and 17-allylamino-17-demethoxygeldanamycin potentiate the invitro and in vivo radiation response of cervical tumorcells via the heat shock protein 90-mediated intracellular signaling andcytotoxicity. Cancer Res 2003,63(24):8984-8995.PubMed
- Machida H, Matsumoto Y, Shirai M, Kubota N: Geldanamycin, an inhibitor of Hsp90, sensitizes human tumour cells toradiation. Int J Radiat Biol 2003, 79: 973-980. 10.1080/09553000310001626135View ArticlePubMed
- Dote H, Burgan WE, Camphausen K, Tofilon PJ: Inhibition of hsp90 compromises the DNA damage response to radiation. Cancer Res 2006, 66: 9211-9220. 10.1158/0008-5472.CAN-06-2181View ArticlePubMed
- Bull EE, Dote H, Brady KJ: Enhanced tumor cell radiosensitivity and abrogation of G2 and S phase arrestby the Hsp90 inhibitor17-(dimethylaminoethylamino)-17-demethoxygeldanamycin. Clin Cancer Res 2004, 10: 8077-8084. 10.1158/1078-0432.CCR-04-1212View ArticlePubMed
- Sakurai H, Kitamoto Y, Saitoh J: Attenuation of chronic thermotolerance by KNK437, a benzylidene lactamcompound, enhances thermal radiosensitization in mild temperaturehyperthermia combined with low dose-rate irradiation. Int J Radiat Biol 2005, 81: 711-718. 10.1080/09553000500448172View ArticlePubMed
- Ohnishi K, Yokota S, Takahashi A, Ohnishi T: Induction of radiation resistance by a heat shock protein inhibitor, KNK437,in human glioblastoma cells. Int J Radiat Biol 2006,82(8):569-575. 10.1080/09553000600876645View ArticlePubMed
- Vitale I, Galluzzi L, Castedo M, Kroemer G: Mitotic catastrophe: a mechanism for avoiding genomic instability. Nat Rev Mol Cell Biol 2011, 12: 385-392.View ArticlePubMed
- Galluzzi L, Vitale I, Abrams JM: Molecular definitions of cell death subroutines: recommendations of theNomenclature Committee on Cell Death 2012. Cell Death Differ 2012, 19: 107-120. 10.1038/cdd.2011.96View ArticlePubMedPubMed Central
- Sekhar KR, Sonar VN, Muthusamy V: Novel chemical enhancers of heat shock increase thermal radiosensitizationthrough a mitotic catastrophe pathway. Cancer Res 2007,67(2):695-701. 10.1158/0008-5472.CAN-06-3212View ArticlePubMed
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